Trypanosoma brucei is the cause of African sleeping sickness and causes a fatal wasting disease of domestic animals. During mammalian infection, the parasite differentiates in the bloodstream from a slender into a stumpy trypanosome. Upon ingestion by the tsetse fly, stumpies differentiate into procyclics in the insect midgut. Differentiation entails survival in two vastly different hosts and a shift in metabolism from glycolysis to cytochrome mediated respiration. While studying protein processing by trypanosomal cysteine protease activity when slender trypanosomes differentiate into stumpies. In a laboratory strain of T. brucei that does not differentiate, there is no increase in protease activity, unless it is exposed to difluoromethylornithine (DFMO), which causes polyamine depletion. The protease has a substrate preference that is most similar to mammalian cathepsin L and is contained in the trypanosomal lysosome. Fast protein liquid chromatography (FPLC) anion exchange, hydrophobic interaction and sizing chromatography conditions that result in purification of active enzyme have been determined. The purified trypanosomal protease cDNA expression library. The gene and its flanking sequences will be cloned and characterized by restriction mapping and sequencing. This will allow comparison of the trypanosomal enxzyme with other known cysteine proteases. The synthesis and turnover rates of the protease and its mRNA will be compared in the different life cycle stages and under varying degrees of polyamine depletion. If expression of the protease is not regulated at the transcriptional level, the effect of polyamine levels on translation of the protease mRNA will be determined. These studies will define the level at which the expression of this protease is regulated. The induction of the trypanosomal protease by polyamine depletion is unique for an enzyme outside of the polyamine synthetic pathway. These studies will, therefore, detail a novel relationship between polyamine depletion and protease induction. Furthermore, since DFMO is likely to become a clinically important anti-trypanosomal agent, it is important to define its metabolic effects. Since the protease of some pathogens have been implicated as virulence factors, determining the regulatory mechanism of protease expression may suggest methods for interference with the virulence and life cycle T. brucei.